The standard, quantitative determination of neomycin activity by the agar diffusion method requires an 18 to 24 h incubation time. To reduce incubation time, an alternative method using triphenyltetrazolium chloride has been developed. The inhibition zone appears much earlier; hence, an effort was made to standardize it. This indicator develops a physical response, related to the biological one, after a determined period. A Staphylococcus epidermidis suspension inoculated into solid medium at a concentration low enough that growth is not visible can reduce the dye to formazan. There is enough contrast to read the inhibition zone optically after a 7 h incubation. There is no significant difference between standard curves for 7 and 24 h incubations. Comparative assays for some pharmaceutical drugs containing neomycin in different forms show that it is possible to reduce incubation time. This modification is valid because no dispersion of results was detected by statistical analysis, which indicates that they belong to the same population. Thus, a rapid microbiological assay of neomycin has been validated and standardized.